title: Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.
abstract: BACKGROUND
  The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation.
  
  
  OBJECTIVE
  To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells.
  
  
  METHODS
  We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC).
  
  
  RESULTS
  Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals.
  
  
  CONCLUSIONS
  We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence.
authors.affiliation: Division of Psychiatry Research and Psychogeriatric Medicine, University of Zurich, Zurich, Switzerland.
authors.forename: Florian
authors.initials: F
authors.lastname: Riese
authors.forename: Sonja
authors.initials: S
authors.lastname: Grinschgl
authors.forename: Manuel T
authors.initials: MT
authors.lastname: Gersbacher
authors.forename: Natalie
authors.initials: N
authors.lastname: Russi
authors.forename: Christoph
authors.initials: C
authors.lastname: Hock
authors.forename: Roger M
authors.initials: RM
authors.lastname: Nitsch
authors.forename: Uwe
authors.initials: U
authors.lastname: Konietzko
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D002467:Cell Nucleus / Q000502:physiology*; D005434:Flow Cytometry; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000235:genetics* / Q000502:physiology; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000074443:Lysine Acetyltransferase 5; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000235:genetics* / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C091042:APBB1 protein, human; C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D051548:Histone Acetyltransferases; C098489:KAT5 protein, human; D000074443:Lysine Acetyltransferase 5; D053829:Amyloid Precursor Protein Secretases